Position-specific carbon isotope analysis of serine by gas chromatography/Orbitrap mass spectrometry, and an application to plant metabolism.

RATIONALE: Position-specific 13 C/12 C ratios within amino acids remain largely unexplored in environmental samples due to methodological limitations. We hypothesized that natural-abundance isotope patterns in serine may serve as a proxy for plant metabolic fluxes including photorespiration. Here we describe an Orbitrap method optimized for the position-specific carbon isotope analysis of serine to test our hypothesis and discuss the generalizability of this method to other amino acids. METHODS: Position-specific carbon isotope ratios of serine were measured using a Thermo Scientific™ Q Exactive™ GC Orbitrap™. Amino acids were hydrolyzed from Arabidopsis biomass, purified from potential matrix interferences, and derivatized alongside standards. Derivatized serine (N,O-bis(trifluoroacetyl)methyl ester) was isolated using gas chromatography, trapped in a reservoir, and purged into the electron ionization source over tens of minutes, producing fragment ions containing different combinations of atoms from the serine-derivative molecule. The 13 C/12 C ratios of fragments with monoisotopic masses of 110.0217, 138.0166, and 165.0037 Da were monitored in the mass analyzer and used to calculate position-specific δ13 C values relative to a working standard. RESULTS: This methodology constrains position-specific δ13 C values for nanomole amounts of serine isolated from chemically complex mixtures. The δ13 C values of fragment ions of serine were characterized with ≤1‰ precisions, leading to propagated standard errors of 0.7-5‰ for each carbon position. Position-specific δ13 C values differed by up to ca 28 ± 5‰ between serine molecules hydrolyzed from plants grown under contrasting pCO2 , selected to promote different fluxes through photosynthesis and photorespiration. The method was validated using pure serine standards characterized offline. CONCLUSIONS: This study presents the first Orbitrap-based measurements of natural-abundance, position-specific carbon isotope variation in an amino acid isolated from a biological matrix. We present a method for the precise characterization of isotope ratios in serine and propose applications probing metabolism in plants. We discuss the potential for extending these approaches to other amino acids, paving the way for novel applications.

Reaction Energetics and 13C Fractionation of Alanine Transamination in the Aqueous and Gas Phases.

The alanine transaminase enzyme catalyzes the transfer of an amino group from alanine to α-ketoglutarate to produce pyruvate and glutamate. Isotope fractionation factors (IFFs) for the reaction +H3NCH(CH3)COO- + -OOCCH2CH2C(O)COO- ↔ CH3C(O)COO- + +H3NCH(CH2CH2COO-)COO- (zwitterionic neutral alanine + doubly deprotonated α-ketoglutarate ↔ pyruvate + zwitterionic glutamate anion) were calculated from the partition functions of explicitly and implicitly solvated molecules at 298 K. Calculations were done for alanine (noncharge separated, zwitterion, deprotonated), pyruvic acid (neutral, deprotonated), glutamic acid (noncharge separated, zwitterion, deprotonated, doubly deprotonated), and α-ketoglutaric acid (neutral, deprotonated, doubly deprotonated). The computational results, calculated from gas phase- and aqueous-optimized clusters with explicit H2O molecules at the MP2/aug-cc-pVDZ and MP2/aug-cc-pVDZ/COSMO levels, respectively, predict that substitution of 13C at the C2 position of alanine and pyruvic acid and their various forms leads to the C2 position of pyruvic acid/pyruvate being enriched in 13C/12C ratio by 9‰. Simpler approaches that estimate the IFFs based solely on changes in the zero-point energies (ZPEs) are consistent with the higher-level model. ZPE-based IFFs calculated for simple analogues formaldehyde and methylamine (analogous to the C2 positions of pyruvate and alanine, respectively) predict a 13C enrichment in formaldehyde of 7-8‰ at the MP2/aug-cc-pVDZ and aug-cc-pVTZ levels. A simple predictive model using canonical functional group frequencies and reduced masses for 13C exchange between R2C═O and R2CH-NH2 predicted enrichment in R2C═O that is too large by a factor of two but is qualitatively accurate compared with the more sophisticated models. Our models are all in agreement with the expectation that pyruvate and formaldehyde will be preferentially enriched in 13C because of the strength of their >C═O bond relative to that of ≡C-NH2 in alanine and methylamine. 13C/12C substitution is also modeled at the methyl and carboxylic acid sites of alanine and pyruvic acid, respectively.